Tumor Antigens BFA5 for Prevention and/or Treatment of Cancer

ABSTRACT

The present invention relates to a nucleic acid encoding a polypeptide and the use of the nucleic acid or polypeptide in preventing and/or treating cancer. In particular, the invention relates to improved vectors for the insertion and expression of foreign genes encoding tumor antigens for use in immunotherapeutic treatment of cancer.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/462,945 filed Apr. 15, 2003.

FIELD OF THE INVENTION

The present invention relates to a nucleic acid encoding a polypeptideand the use of the nucleic acid or polypeptide in preventing and/ortreating cancer. In particular, the invention to relates to improvedvectors for the insertion and expression of foreign genes encoding tumorantigens for use in immunotherapeutic treatment of cancer.

BACKGROUND OF THE INVENTION

There has been tremendous increase in last few years in the developmentof cancer vaccines with Tumour-associated antigens (TAAs) due to thegreat advances in identification of molecules based on the expressionprofiling on primary tumours and normal cells with the help of severaltechniques such as high density microarray, SEREX, immunohistochemistry(IHC), RT-PCR, in-situ hybridization (ISH) and laser capture microscopy(Rosenberg, Immunity, 1999; Sgroi et al, 1999, Schena et al, 1995,Offring a et al, 2000). The TAAs are antigens expressed orover-expressed by tumour cells and could be specific to one or severaltumours for example CEA antigen is expressed in colorectal, breast andlung cancers. Sgroi et al. (1999) identified several genesdifferentially expressed in invasive and metastatic carcinoma cells withcombined use of laser capture microdissection and cDNA microarrays.Several delivery systems like DNA or viruses could be used fortherapeutic vaccination against human cancers (Bonnet et al, 2000) andcan elicit immune responses and also break immune tolerance againstTAAs. Tumour cells can be rendered more immunogenic by insertingtransgenes encoding T cell co-stimulatory molecules such as B7.1 orcytokines such as IFN-γ, IL2, or GM-CSF, among others. Co-expression ofa TAA and a cytokine or a co-stimulatory molecule has also been shown tobe useful in developing effective therapeutic vaccines (Hodge et al, 95,Bronze et al, 1995, Chamberlain et al, 1996).

There is a need in the art for reagents and methodologies useful instimulating an immune response to prevent or treat cancers. The presentinvention provides such reagents and methodologies which overcome manyof the difficulties encountered by others in attempting to treat cancer.

SUMMARY OF THE INVENTION

The present invention provides an immunogenic target for administrationto a patient to prevent and/or treat cancer. In particular, theimmunogenic target is a tumor antigen (“TA”) and/or anangiogenesis-associated antigen (“AA”). In one embodiment, theimmunogenic target is encoded by SEQ ID NO.: 5 or has the amino acidsequence of SEQ ID NO.: 6. In certain embodiments, the TA and/or AA areadministered to a patient as a nucleic acid contained within a plasmidor other delivery vector, such as a recombinant virus. The TA and/or AAmay also be administered in combination with additional tumor antigens(i.e., SEQ ID NOS.: 1-4) and/or an immune stimulator, such as aco-stimulatory molecule or adjuvant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. BFA4 cDNA sequence.

FIG. 2. BFA4 amino acid sequence.

FIG. 3. BCY1 nucleotide (A) and amino acid (B) sequences.

FIG. 4. BFA5 cDNA sequence.

FIG. 5. BFA5 amino acid sequence.

DETAILED DESCRIPTION

The present invention provides reagents and methodologies useful fortreating and/or preventing cancer. All references cited within thisapplication are incorporated by reference.

In one embodiment, the present invention relates to the induction orenhancement of an immune response against one or more tumor antigens(“TA”) to prevent and/or treat cancer. In certain embodiments, one ormore TAs may be combined. In preferred embodiments, the immune responseresults from expression of a TA in a host cell following administrationof a nucleic acid vector encoding the tumor antigen or the tumor antigenitself in the form of a peptide or polypeptide, for example.

As used herein, an “antigen” is a molecule such as a polypeptide or aportion thereof that produces an immune response in a host to whom theantigen has been administered. The immune response may include theproduction of antibodies that bind to at least one epitope of theantigen and/or the generation of a cellular immune response againstcells expressing an epitope of the antigen. The response may be anenhancement of a current immune response by, for example, causingincreased antibody production, production of antibodies with increasedaffinity for the antigen, or an increased or more effective cellularresponse (i.e., increased T cells or T cells with higher anti-tumoractivity). An antigen that produces an immune response may alternativelybe referred to as being immunogenic or as an immunogen. In describingthe present invention, a TA may be referred to as an “immunogenictarget”.

TA includes both tumor-associated antigens (TAAs) and tumor-specificantigens (TSAs), where a cancerous cell is the source of the antigen. ATAA is an antigen that is expressed on the surface of a tumor cell inhigher amounts than is observed on normal cells or an antigen that isexpressed on normal cells during fetal development. A TSA is an antigenthat is unique to tumor cells and is not expressed on normal cells. TAfurther includes TAAs or TSAs, antigenic fragments thereof, and modifiedversions that retain their antigenicity.

TAs are typically classified into five categories according to theirexpression pattern, function, or genetic origin: cancer-testis (CT)antigens (i.e., MAGE, NY-ESO-1); melanocyte differentiation antigens(i.e., Melan A/MART-1, tyrosinase, gp100); mutational antigens (i.e.,MUM-1, p53, CDK-4); overexpressed ‘self’ antigens (i.e., HER-2/neu,p53); and, viral antigens (i.e., HPV, EBV). For the purposes ofpracticing the present invention, a suitable TA is any TA that inducesor enhances an anti-tumor immune response in a host in whom the TA isexpressed. Suitable TAs include, for example, gp100 (Cox et al.,Science, 264:716-719 (1994)), MART-1/Melan A (Kawakami et al., J. Exp.Med., 180:347-352 (1994)), gp75 (TRP-1) (Wang et al., J. Exp. Med.,186:1131-1140 (1996)), tyrosinase (Wolfel et al., Eur. J. Immunol.,24:759-764 (1994); WO 200175117; WO 200175016; WO 200175007), NY-ESO-1(WO 98/14464; WO 99/18206), melanoma proteoglycan (Hellstrom et al., J.Immunol., 130:1467-1472 (1983)), MAGE family antigens (i.e.,MAGE-1,2,3,4,6,12,51; Van der Bruggen et al., Science, 254:1643-1647(1991); U.S. Pat. Nos. 6,235,525; CN 1319611), BAGE family antigens(Boel et al., Immunity, 2:167-175 (1995)), GAGE family antigens (i.e.,GAGE-1,2; Van den Eynde et al., J. Exp. Med., 182:689-698 (1995); U.S.Pat. No. 6,013,765), RAGE family antigens (i.e., RAGE-1; Gaugler et al.,Immunogenetics, 44:323-330 (1996); U.S. Pat. No. 5,939,526),N-acetylglucosaminyltransferase-V (Guilloux et al., J. Exp. Med.,183:1173-1183 (1996)), p15 (Robbins et al., J. Immunol. 154:5944-5950(1995)), β-catenin (Robbins et al., J. Exp. Med., 183:1185-1192 (1996)),MUM-1 (Coulie et al., Proc. Natl. Acad. Sci. USA, 92:7976-7980 (1995)),cyclin dependent kinase-4 (CDK4) (Wolfel et al., Science, 269:1281-1284(1995)), p21-ras (Fossum et al., Int. J. Cancer, 56:40-45 (1994)),BCR-abl (Bocchia et al., Blood, 85:2680-2684 (1995)), p53 (Theobald etal., Proc. Natl. Acad. Sci. USA, 92:11993-11997 (1995)), p185 HER2/neu(erb-B1; Fisk et al., J. Exp. Med., 181:2109-2117 (1995)), epidermalgrowth factor receptor (EGFR) (Harris et al., Breast Cancer Res. Treat,29:1-2 (1994)), carcinoembryonic antigens (CEA) (Kwong et al., J. Natl.Cancer Inst., 85:982-990 (1995) U.S. Pat. Nos. 5,756,103; 5,274,087;5,571,710; 6,071,716; 5,698,530; 6,045,802; EP 263933; EP 346710; and,EP 784483); carcinoma-associated mutated mucins (i.e., MUC-1 geneproducts; Jerome et al., J. Immunol., 151:1654-1662 (1993)); EBNA geneproducts of EBV (i.e., EBNA-1; Rickinson et al., Cancer Surveys,13:53-80 (1992)); E7, E6 proteins of human papillomavirus (Ressing etal., J. Immunol, 154:5934-5943 (1995)); prostate specific antigen (PSA;Xue et al., The Prostate, 30:73-78 (1997)); prostate specific membraneantigen (PSMA; Israeli, et al., Cancer Res., 54:1807-1811 (1994));idiotypic epitopes or antigens, for example, immunoglobulin idiotypes orT cell receptor idiotypes (Chen et al., J. Immunol., 153:4775-4787(1994)); KSA (U.S. Pat. No. 5,348,887), kinesin 2 (Dietz, et al. BiochemBiophys Res Commun 2000 Sep. 7; 275(3):731-8), HIP-55, TGFβ-1anti-apoptotic factor (Toomey, et al. Br J Biomed Sci 2001;58(3):177-83), tumor protein D52 (Bryne J. A., et al., Genomics,35:523-532 (1996)), H1FT, NY-BR-1 (WO 01/47959), NY-BR-62, NY-BR-75,NY-BR-85, NY-BR-87, NY-BR-96 (Scanlan, M. Serologic and BioinformaticApproaches to the Identification of Human Tumor Antigens, in CancerVaccines 2000, Cancer Research Institute, New York, N.Y.), BFA4 (SEQ IDNOS.: 1 and 2), BCY1 (SEQ ID NOS.: 3 and 4), and BFA5 (SEQ ID NOS.: 5and 6) including “wild-type” (i.e., normally encoded by the genome,naturally-occurring), modified, and mutated versions as well as otherfragments and derivatives thereof. Any of these TAs may be utilizedalone or in combination with one another in a co-immunization protocol.

In certain cases, it may be beneficial to co-immunize patients with bothTA and other antigens, such as angiogenesis-associated antigens (“AA”).An AA is an immunogenic molecule (i.e., peptide, polypeptide) associatedwith cells involved in the induction and/or continued development ofblood vessels. For example, an AA may be expressed on an endothelialcell (“EC”), which is a primary structural component of blood vessels.For treatment of cancer, it is preferred that that the AA be foundwithin or near blood vessels that supply a tumor. Immunization of apatient against an AA preferably results in an anti-AA immune responsewhereby angiogenic processes that occur near or within tumors areprevented and/or inhibited.

Exemplary AAs include, for example, vascular endothelial growth factor(i.e., VEGF; Bernardini, et al. J. Urol., 2001, 166(4): 1275-9; Starnes,et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23; Dias, et al.Blood, 2002, 99: 2179-2184), the VEGF receptor (i.e., VEGF-R, fik-1/KDR;Starnes, et al. J. Thorac. Cardiovasc. Surg., 2001, 122(3): 518-23), EPHreceptors (i.e., EPHA2; Gerety, et al. 1999, Cell, 4: 403-414),epidermal growth factor receptor (i.e., EGFR; Ciardeillo, et al. Clin.Cancer Res., 2001, 7(10): 2958-70), basic fibroblast growth factor(i.e., bFGF; Davidson, et al. Clin. Exp. Metastasis 2000,18(6): 501-7;Poon, et al. Am J. Surg., 2001, 182(3):298-304), platelet-derived cellgrowth factor (i.e., PDGF-B), platelet-derived endothelial cell growthfactor (PD-ECGF; Hong, et al. J. Mol. Med., 2001, 8(2):141-8),transforming growth factors (i.e., TGF-α; Hong, et al. J. Mol. Med.,2001, 8(2):141-8), endoglin (Balza, et al. Int. J. Cancer, 2001, 94:579-585), Id proteins (Benezra, R. Trends Cardiovasc. Med., 2001,11(6):237-41), proteases such as uPA, uPAR, and matrixmetalloproteinases (MMP-2, MMP-9; Djonov, et al. J. Pathol., 2001,195(2):147-55), nitric oxide synthase (Am. J. Ophthalmol., 2001,132(4):551-6), aminopeptidase (Rouslhati, E. Nature Cancer, 2: 84-90,2002), thrombospondins (i.e., TSP-1, TSP-2; Alvarez, et al. Gynecol.Oncol., 2001, 82(2):273-8; Seki, et to al. Int. J. Oncol., 2001,19(2):305-10), k-ras (Zhang, et al. Cancer Res., 2001, 61(16):6050-4),Wnt (Zhang, et al. Cancer Res., 2001, 61(16):6050-4), cyclin-dependentkinases (CDKs; Drug Resist. Updat. 2000, 3(2):83-88), microtubules(Timar, et al. 2001. Path. Oncol. Res., 7(2): 85-94), heat shockproteins (i.e., HSP90 (Timar, supra)), heparin-binding factors (i.e.,heparinase; Gohji, et al. Int. J. Cancer, 2001, 95(5):295-301),synthases (i.e., ATP synthase, thymidilate synthase), collagenreceptors, integrins (i.e., ανβ3, ανβ5, α1β1, α2β1, α5β1), the surfaceproteolglycan NG2, AAC2-1 (SEQ ID NO.:1), or AAC2-2 (SEQ ID NO.:2),among others, including “wild-type” (i.e., normally encoded by thegenome, naturally-occurring), modified, mutated versions as well asother fragments and derivatives thereof. Any of these targets may besuitable in practicing the present invention, either alone or incombination with one another or with other agents.

In certain embodiments, a nucleic acid molecule encoding an immunogenictarget is utilized. The nucleic acid molecule may comprise or consist ofa nucleotide sequence encoding one or more immunogenic targets, orfragments or derivatives thereof, such as that contained in a DNA insertin an ATCC Deposit. The term “nucleic acid sequence” or “nucleic acidmolecule” refers to a DNA or RNA sequence. The term encompassesmolecules formed from any of the known base analogs of DNA and RNA suchas, but not limited to 4-acetylcytosine, 8-hydroxy-N6-methyladenosine,aziridinyl-cytosine, pseudoisocytosine, 5-(carboxyhydroxylmethyl)uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxy-methylaminomethyluracil, dihydrouracil, inosine,N6-iso-pentenyladenine, 1-methyladenine, 1-methylpseudouracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyamino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonyl-methyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil,queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouraoil,4-thiouracil, 5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine, among others.

An isolated nucleic acid molecule is one that: (1) is separated from atleast about 50 percent of proteins, lipids, carbohydrates, or othermaterials with which it is naturally found when total nucleic acid isisolated from the source cells; (2) is not linked to all or a portion ofa polynucleotide to which the nucleic acid molecule is linked in nature;(3) is operably linked to a polynucleotide which it is not linked to innature; and/or, (4) does not occur in nature as part of a largerpolynucleotide sequence. Preferably, the isolated nucleic acid moleculeof the present invention is substantially free from any othercontaminating nucleic acid molecule(s) or other contaminants that arefound in its natural environment that would interfere with its use inpolypeptide production or its therapeutic, diagnostic, prophylactic orresearch use. As used herein, the term “naturally occurring” or “native”or “naturally found” when used in connection is with biologicalmaterials such as nucleic acid molecules, polypeptides, host cells, andthe like, refers to materials which are found in nature withoutmanipulation by man. Similarly, “non-naturally occurring” or“non-native” as used herein refers to a material that is not found innature or that has been structurally modified or synthesized by man.

The identity of two or more nucleic acid or polypeptide molecules isdetermined by comparing the sequences. As known in the art, “identity”means the degree of sequence relatedness between nucleic acid moleculesor polypeptides as determined by the match between the units making upthe molecules (i.e., nucleotides or amino acid residues). Identitymeasures the percent of identical matches between the smaller of two ormore sequences with gap alignments (if any) addressed by a particularmathematical model or computer program (i.e., an algorithm). Identitybetween nucleic acid sequences may also be determined by the ability ofthe related sequence to hybridize to the nucleic acid sequence orisolated nucleic acid molecule. In defining such sequences, the term“highly stringent conditions” and “moderately stringent conditions”refer to procedures that permit hybridization of nucleic acid strandswhose sequences are complementary, and to exclude hybridization ofsignificantly mismatched nucleic acids. Examples of “highly stringentconditions” for hybridization and washing are 0.015 M sodium chloride,0.0015 M sodium citrate at 65-68° C. or 0.015 M sodium chloride, 0.0015M sodium citrate, and 50% formamide at 42° C. (see, for example,Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual(2nd ed., Cold Spring Harbor Laboratory, 1989); Anderson et al., NucleicAcid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited)). Theterm “moderately stringent conditions” refers to conditions under whicha DNA duplex with a greater degree of base pair mismatching than couldoccur under “highly stringent conditions” is able to form. Exemplarymoderately stringent conditions are 0.015 M sodium chloride, 0.0015 Msodium citrate at 50-65° C. or 0.015 M sodium chloride, 0.0015 M sodiumcitrate, and 20% formamide at 37-50° C. By way of example, moderatelystringent conditions of 50° C. in 0.015 M sodium ion will allow about a21% mismatch. During hybridization, other agents may be included in thehybridization and washing buffers for the purpose of reducingnon-specific and/or background hybridization. Examples are 0.1% bovineserum albumin, 0.1% polyvinyl-pyrrolidone, 0.1% sodium pyrophosphate,0.1% sodium dodecylsulfate, NaDodSO₄, (SDS), ficoll, Denhardt'ssolution, sonicated salmon sperm DNA (or another non-complementary DNA),and dextran sulfate, although other suitable agents can also be used.The concentration and types of these additives can be changed withoutsubstantially affecting the stringency of the hybridization conditions.Hybridization experiments are usually carried out at pH 6.8-7.4;however, at typical ionic strength conditions, the rate of hybridizationis nearly independent of pH.

In certain embodiments of the present invention, vectors are used totransfer a nucleic acid sequence encoding a polypeptide to a cell. Avector is any molecule used to transfer a nucleic acid sequence to ahost cell. In certain cases, an expression vector is utilized. Anexpression vector is a nucleic acid molecule that is suitable fortransformation of a host cell and contains nucleic acid sequences thatdirect and/or control the expression of the transferred nucleic acidsequences. Expression includes, but is not limited to, processes such astranscription, translation, and splicing, if introns are present.Expression vectors typically comprise one or more flanking sequencesoperably linked to a heterologous nucleic acid sequence encoding apolypeptide. Flanking sequences may be homologous (i.e., from the samespecies and/or strain as the host cell), heterologous (i.e., from aspecies other than the host cell species or strain), hybrid (i.e., acombination of flanking sequences from more than one source), orsynthetic, for example.

A flanking sequence is preferably capable of effecting the replication,transcription and/or translation of the coding sequence and is operablylinked to a coding sequence. As used herein, the term operably linkedrefers to a linkage of polynucleotide elements in a functionalrelationship. For instance, a promoter or enhancer is operably linked toa coding sequence if it affects the transcription of the codingsequence. However, a flanking sequence need not necessarily becontiguous with the coding sequence, so long as it functions correctly.Thus, for example, intervening untranslated yet transcribed sequencescan be present between a promoter sequence and the coding sequence andthe promoter sequence may still be considered operably linked to thecoding sequence. Similarly, an enhancer sequence may be located upstreamor downstream from the coding sequence and affect transcription of thesequence.

In certain embodiments, it is preferred that the flanking sequence is atranscriptional regulatory region that drives high-level gene expressionin the target cell. The transcriptional regulatory region may comprise,for example, a promoter, enhancer, silencer, repressor element, orcombinations thereof. The transcriptional regulatory region may beeither constitutive, tissue-specific, cell-type specific (i.e., theregion is drives higher levels of transcription in a one type of tissueor cell as compared to another), or regulatable (i.e., responsive tointeraction with a compound). The source of a transcriptional regulatoryregion may be any prokaryotic or eukaryotic organism, any vertebrate orinvertebrate organism, or any plant, provided that the flanking sequencefunctions in a cell by causing transcription of a nucleic acid withinthat cell. A wide variety of transcriptional regulatory regions may beutilized in practicing the present invention.

Suitable transcriptional regulatory regions include, for example, theCMV promoter (i.e., the CMV-immediate early promoter); promoters fromeukaryotic genes (i.e., the estrogen-inducible chicken ovalbumin gene,the interferon genes, the gluco-corticoid-inducible tyrosineaminotransferase gene, and the thymidine kinase gene); and the majorearly and late adenovirus gene promoters; the SV40 early promoter region(Bernoist and Chambon, 1981, Nature 290:304-10); the promoter containedin the 3′ long terminal repeat (LTR) of Rous sarcoma virus (RSV)(Yamamoto, et al., 1980, Cell 22:787-97); the herpes simplex virusthymidine kinase (HSV-TK) promoter (Wagner et al., 1981, Proc. Natl.Acad. Sci. U.S.A. 78:1444-45); the regulatory sequences of themetallothionine gene (Brirster et al., 1982, Nature 296:39-42);prokaryotic expression vectors such as the beta-lactamase promoter(Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. U.S.A.,75:3727-31); or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad.Sci. U.S.A., 80:21-25). Tissue- and/or cell-type specifictranscriptional control regions include, for example, the elastase Igene control region which is active in pancreatic acinar cells (Swift etal., 1984, Cell 38:639-46; Omitz et al., 1986, Cold Spring Harbor Symp.Quant. Biol. 50:399-409 (1986); MacDonald, 1987, Hepatology 7:425-515);the insulin gene control region which is active in pancreatic beta cells(Hanahan, 1985, Nature 315:115-22); the immunoglobulin gene controlregion which is active in lymphoid cells (Grosschedl et al., 1984, Cell38:647-58; Adames et al., 1985, Nature 318:533-38; Alexander et al.,1987, Mol. Cell. Biol., 7:1436-44); the mouse mammary tumor viruscontrol region in testicular, breast, lymphoid and mast cells (Leder etal., 1986, Cell 45:485-95); the albumin gene control region in liver(Pinkert et al., 1987, Genes and Devel. 1:268-76); thealpha-feto-protein gene control region in liver (Krumlauf et al., 1985,Mol. Cell. Biol., 5:1639-48; Hammer et al., 1987, Science 235:53-58);the alpha 1-antitrypsin gene control region in liver (Kelsey et al.,1987, Genes and Devel. 1:161-71); the beta-globin gene control region inmyeloid cells (Mogram et al., 1985, Nature 315:338-40; Kollias et al.,1986, Cell 46:89-94); the myelin basic protein gene control region inoligodendrocyte cells in the brain (Readhead et al., 1987, Cell48:703-12); the myosin light chain-2 gene control region in skeletalmuscle (Sani, 1985, Nature 314:283-86); the gonadotropic releasinghormone gene control region in the hypothalamus (Mason et al., 1986,Science 234:1372-78), and the tyrosinase promoter in melanoma cells(Hart, I. Semin Oncol 1996 February; 23(1):154-8; Siders, et al. CancerGene Ther 1998 September-October; 5(5):281-91), among others. Induciblepromoters that are activated in the presence of a certain compound orcondition such as light, heat, radiation, tetracycline, or heat shockproteins, for example, may also be utilized (see, for example, WO00/10612). Other suitable promoters are known in the art.

As described above, enhancers may also be suitable flanking sequences.Enhancers are cis-acting elements of DNA, usually about 10-300 by inlength, that act on the promoter to increase transcription. Enhancersare typically orientation- and position-independent, having beenidentified both 5′ and 3′ to controlled coding sequences. Severalenhancer sequences available from mammalian genes are known (i.e.,globin, elastase, albumin, alpha-feto-protein and insulin). Similarly,the SV40 enhancer, the cytomegalovirus early promoter enhancer, thepolyoma enhancer, and adenovirus enhancers are useful with eukaryoticpromoter sequences. While an enhancer may be spliced into the vector ata position 5′ or 3′ to nucleic acid coding sequence, it is typicallylocated at a site 5′ from the promoter. Other suitable enhancers areknown in the art, and would be applicable to the present invention.

While preparing reagents of the present invention, cells may need to betransfected or transformed. Transfection refers to the uptake of foreignor exogenous DNA by a cell, and a cell has been transfected when theexogenous DNA has been introduced inside the cell membrane. A number oftransfection techniques are well known in the art (i.e., Graham et al.,1973, Virology 52:456; Sambrook et al., Molecular Cloning, A LaboratoryManual (Cold Spring Harbor Laboratories, 1989); Davis et al., BasicMethods in Molecular Biology (Elsevier, 1986); and Chu et al., 1981,Gene 13:197). Such techniques can be used to introduce one or moreexogenous DNA moieties into suitable host cells.

In certain embodiments, it is preferred that transfection of a cellresults in transformation of that cell. A cell is transformed when thereis a change in a characteristic of the cell, being transformed when ithas been modified to contain a new nucleic acid. Following transfection,the transfected nucleic acid may recombine with that of the cell byphysically integrating into a chromosome of the cell, may be maintainedtransiently as an episomal element without being replicated, or mayreplicate independently as a plasmid. A cell is stably transformed whenthe nucleic acid is replicated with the division of the cell.

The present invention further provides isolated immunogenic targets inpolypeptide form. A polypeptide is considered isolated where it: (1) hasbeen separated from at least about 50 percent of polynucleotides,lipids, carbohydrates, or other materials with which it is naturallyfound when isolated from the source cell; (2) is not linked (by covalentor noncovalent interaction) to all or a portion of a polypeptide towhich the “isolated polypeptide” is linked in nature; (3) is operablylinked (by covalent or noncovalent interaction) to a polypeptide withwhich it is not linked in nature; or, (4) does not occur in nature.Preferably, the isolated polypeptide is substantially free from anyother contaminating polypeptides or other contaminants that are found inits natural environment that would interfere with its therapeutic,diagnostic, prophylactic or research use.

Immunogenic target polypeptides may be mature polypeptides, as definedherein, and may or may not have an amino terminal methionine residue,depending on the method by which they are prepared. Further contemplatedare related polypeptides such as, for example, fragments, variants(i.e., allelic, splice), orthologs, homologues, and derivatives, forexample, that possess at least one characteristic or activity (i.e.,activity, antigenicity) of the immunogenic target. Also related arepeptides, which refers to a series of contiguous amino acid residueshaving a sequence corresponding to at least a portion of the polypeptidefrom which its sequence is derived. In preferred embodiments, thepeptide comprises about 5-10 amino acids, 10-15 amino acids, 15-20 aminoacids, 20-30 amino acids, or 30-50 amino acids. In a more preferredembodiment, a peptide comprises 9-12 amino acids, suitable forpresentation upon Class I MHC molecules, for example.

A fragment of a nucleic acid or polypeptide comprises a truncation ofthe sequence (i.e., nucleic acid or polypeptide) at the amino terminus(with or without a leader sequence) and/or the carboxy terminus.Fragments may also include variants (i.e., allelic, splice), orthologs,homologues, and other variants having one or more amino acid additionsor substitutions or internal deletions as compared to the parentalsequence. In preferred embodiments, truncations and/or deletionscomprise about 10 amino acids, 20 amino acids, 30 amino acids, 40 aminoacids, 50 amino acids, or more. The polypeptide fragments so producedwill comprise about 10 amino acids, 25 amino acids, 30 amino acids, 40amino acids, 50 amino acids, 60 amino acids, 70 amino acids, or more.Such polypeptide fragments may optionally comprise an amino terminalmethionine residue. It will be appreciated that such fragments can beused, for example, to generate antibodies or cellular immune responsesto immunogenic target polypeptides.

A variant is a sequence having one or more sequence substitutions,deletions, and/or additions as compared to the subject sequence.Variants may be naturally occurring or artificially constructed. Suchvariants may be prepared from the corresponding nucleic acid molecules.In preferred embodiments, the variants have from 1 to 3, or from 1 to 5,or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, orfrom 1 to 30, or from Ito 40, or from 1 to 50, or more than 50 aminoacid substitutions, insertions, additions and/or deletions.

An allelic variant is one of several possible naturally-occurringalternate forms of a gene occupying a given locus on a chromosome of anorganism or a population of organisms. A splice variant is a polypeptidegenerated from one of several RNA transcript resulting from splicing ofa primary transcript. An ortholog is a similar nucleic acid orpolypeptide sequence from another species. For example, the mouse andhuman versions of an immunogenic target polypeptide may be consideredorthologs of each other. A derivative of a sequence is one that isderived from a parental sequence those sequences having substitutions,additions, deletions, or chemically modified variants. Variants may alsoinclude fusion proteins, which refers to the fusion of one or more firstsequences (such as a peptide) at the amino or carboxy terminus of atleast one other sequence (such as a heterologous peptide).

“Similarity” is a concept related to identity, except that similarityrefers to a measure of relatedness which includes both identical matchesand conservative substitution matches. If two polypeptide sequenceshave, for example, 1020 identical amino acids, and the remainder are allnon-conservative substitutions, then the percent identity and similaritywould both be 50%. If in the same example, there are five more positionswhere there are conservative substitutions, then the percent identityremains 50%, but the percent similarity would be 75% (15/20). Therefore,in cases where there are conservative substitutions, the percentsimilarity between two polypeptides will be higher than the percentidentity between those two polypeptides.

Substitutions may be conservative, or non-conservative, or anycombination thereof. Conservative amino acid modifications to thesequence of a polypeptide (and the corresponding modifications to theencoding nucleotides) may produce polypeptides having functional andchemical characteristics similar to those of a parental polypeptide. Forexample, a “conservative amino acid substitution” may involve asubstitution of a native amino acid residue with a non-native residuesuch that there is little or no effect on the size, polarity, charge,hydrophobicity, or hydrophilicity of the amino acid residue at thatposition and, in particular, does not result in decreasedimmunogenicity. Suitable conservative amino acid substitutions are shownin Table I.

TABLE I Original Preferred Residues Exemplary SubstitutionsSubstitutions Ala Val, Leu, Ile Val Arg Lys, Gln, Asn Lys Asn Gln GlnAsp Glu Glu Cys Ser, Ala Ser Gln Asn Asn Glu Asp Asp Gly Pro, Ala AlaHis Asn, Gln, Lys, Arg Arg Ile Leu, Val, Met, Ala, Phe, Norleucine LeuLeu Norleucine, Ile, Val, Met, Ala, Phe Ile Lys Arg, 1,4 Diamino-butyricAcid, Gln, Asn Arg Met Leu, Phe, Ile Leu Phe Leu, Val, Ile, Ala, Tyr LeuPro Ala Gly Ser Thr, Ala, Cys Thr Thr Ser Ser Trp Tyr, Phe Tyr Tyr Trp,Phe, Thr, Ser Phe Val Ile, Met, Leu, Phe, Ala, Norleucine Leu

A skilled artisan will be able to determine suitable variants ofpolypeptide using well-known techniques. For identifying suitable areasof the molecule that may be changed without to destroying biologicalactivity (i.e., MHC binding, immunogenicity), one skilled in the art maytarget areas not believed to be important for that activity. Forexample, when similar polypeptides with similar activities from the samespecies or from other species are known, one skilled in the art maycompare the amino acid sequence of a polypeptide to such similarpolypeptides. By performing such analyses, one can identify residues andportions of the molecules that are conserved among similar polypeptides.It will be appreciated that changes in areas of the molecule that arenot conserved relative to such similar polypeptides would be less likelyto adversely affect the biological activity and/or structure of apolypeptide. Similarly, the residues required for binding to MHC areknown, and may be modified to improve binding. However, modificationsresulting in decreased binding to MHC will not be appropriate in mostsituations. One skilled in the art would also know that, even inrelatively conserved regions, one may substitute chemically similaramino acids for the naturally occurring residues while retainingactivity. Therefore, even areas that may be important for biologicalactivity or for structure may be subject to conservative amino acidsubstitutions without destroying the biological activity or withoutadversely affecting the polypeptide structure.

Other preferred polypeptide variants include glycosylation variantswherein the number and/or type of glycosylation sites have been alteredcompared to the subject amino acid sequence. In one embodiment,polypeptide variants comprise a greater or a lesser number of N-linkedglycosylation sites than the subject amino acid sequence. An N-linkedglycosylation site is characterized by the sequence Asn-X-Ser orAsn-X-Thr, wherein the amino acid residue designated as X may be anyamino acid residue except proline. The substitution of amino acidresidues to create this sequence provides a potential new site for theaddition of an N-linked carbohydrate chain. Alternatively, substitutionsthat eliminate this sequence will remove an existing N-linkedcarbohydrate chain. Also provided is a rearrangement of N-linkedcarbohydrate chains wherein one or more N-linked glycosylation sites(typically those that are naturally occurring) are eliminated and one ormore new N-linked sites are created. To affect O-linked glycosylation ofa polypeptide, one would modify serine and/or threonine residues.

Additional preferred variants include cysteine variants, wherein one ormore cysteine residues are deleted or substituted with another aminoacid (e.g., serine) as compared to the subject amino acid sequence set.Cysteine variants are useful when polypeptides must be refolded into abiologically active conformation such as after the isolation ofinsoluble inclusion bodies. Cysteine variants generally have fewercysteine residues than the native protein, and typically have an evennumber to minimize interactions resulting from unpaired cysteines.

In other embodiments, the isolated polypeptides of the current inventioninclude fusion polypeptide segments that assist in purification of thepolypeptides. Fusions can be made either at the amino terminus or at thecarboxy terminus of the subject polypeptide variant thereof. Fusions maybe direct with no linker or adapter molecule or may be through a linkeror adapter molecule. A linker or adapter molecule may be one or moreamino acid residues, typically from about 20 to about 50 amino acidresidues. A linker or adapter molecule may also be designed with acleavage site for a DNA restriction endonuclease or for a protease toallow for the separation of the fused moieties. It will be appreciatedthat once constructed, the fusion polypeptides can be derivatizedaccording to the methods described herein. Suitable fusion segmentsinclude, among others, metal binding domains (e.g., a poly-histidinesegment), immunoglobulin binding domains (i.e., Protein A, Protein G, Tcell, B cell, Fc receptor, or complement protein antibody-bindingdomains), sugar binding domains (e.g., a maltose binding domain), and/ora “tag” domain (i.e., at least a portion of α-galactosidase, a strep tagpeptide, a T7 tag peptide, a FLAG peptide, or other domains that can bepurified using compounds that bind to the domain, such as monoclonalantibodies). This tag is typically fused to the polypeptide uponexpression of the polypeptide, and can serve as a means for affinitypurification of the sequence of interest polypeptide from the host cell.Affinity purification can be accomplished, for example, by columnchromatography using antibodies against the tag as an affinity matrix.Optionally, the tag can subsequently be removed to from the purifiedsequence of interest polypeptide by various means such as using certainpeptidases for cleavage. As described below, fusions may also be madebetween a TA and a co-stimulatory components such as the chemokinesCXC10 (IP-10), CCL7 (MCP-3), or CCL5 (RANTES), for example.

A fusion motif may enhance transport of an immunogenic target to an MHCprocessing compartment, such as the endoplasmic reticulum. Thesesequences, referred to as tranduction or transcytosis sequences, includesequences derived from HIV tat (see Kim et al. 1997 J. Immunol.159:1666), Drosophila antennapedia (see Schutze-Redelmeier et al. 1996J. Immunol. 157:650), or human period-1 protein (hPER1; in particular,SRRHHCRSKAKRSRHH).

In addition, the polypeptide or variant thereof may be fused to ahomologous polypeptide to form a homodimer or to a heterologouspolypeptide to form a heterodimer. Heterologous peptides andpolypeptides include, but are not limited to: an epitope to allow forthe detection and/or isolation of a fusion polypeptide; a transmembranereceptor protein or a portion thereof, such as an extracellular domainor a transmembrane and intracellular domain; a ligand or a portionthereof which binds to a transmembrane receptor protein; an enzyme orportion thereof which is catalytically active; a polypeptide or peptidewhich promotes oligomerization, such as a leucine zipper domain; apolypeptide or peptide which increases stability, such as animmunoglobulin constant region; and a polypeptide which has atherapeutic activity different from the polypeptide or variant thereof.

In certain embodiments, it may be advantageous to combine a nucleic acidsequence encoding an immunogenic target, polypeptide, or derivativethereof with one or more nucleic acid sequences encoding one or moreco-stimulatory component(s) such as cell surface proteins, cytokines orchemokines in a composition of the present invention. The co-stimulatorycomponent may be included in the composition as a polypeptide or as anucleic acid encoding the polypeptide, for example. Suitableco-stimulatory molecules include, for instance, polypeptides that bindmembers of the CD28 family (i.e., CD28, ICOS; Hutloff, et al. Nature1999, 397: 263-265; Peach, et al. J Exp Med 1994, 180: 2049-2058) suchas the CD28 binding polypeptides B7.1 (CD80; Schwartz, 1992; Chen et al,1992; Ellis, et al. J. Immunol., 156(8): 2700-9) and B7.2 (CD86; Ellis,et al. J. Immunol., 156(8): 2700-9); mutated and derivative B7 molecules(WO 00/66162); polypeptides which bind members of the integrin family(i.e., LFA-1 (CD11a/CD18); Sedwick, et al. J Immunol 1999, 162:1367-1375; Wülfing, et al. Science 1998, 282: 2266-2269; Lub, et al.Immunol Today 1995, 16: 479-483) including members of the ICAM family(i.e., ICAM-1, -2 or -3); polypeptides which bind CD2 family members(i.e., CD2, signalling lymphocyte activation molecule (CDw150 or “SLAM”;Aversa, et al. J Immunol 1997, 158: 4036-4044)) such as CD58 (LFA-3; CD2ligand; Davis, et al. Immunol Today 1996, 17: 177-187) or SLAM ligands(Sayos, et al. Nature 1998, 395: 462-469); polypeptides which bind heatstable antigen (HSA or CD24; Zhou, et al. Eur J Immunol 1997, 27:2524-2528); polypeptides which bind to members of the TNF receptor(TNFR) family (i.e., 4-1BB (CD137; Vinay, et al. Semin Immunol 1998, 10:481-489), OX40 (CD134; Weinberg, et al. Semin Immunol 1998, 10: 471-480;Higgins, et al. J Immunol 1999, 162: 486-493), and CD27 (Lens, et al.Semin Immunol 1998, 10: 491-499)) such as 4-1BBL (4-1BB ligand; Vinay,et al. Semin Immunol 1998, 10: 481-48; DeBenedette, et al. J Immunol1997, 158: 551-559), TNFR associated factor-1 (TRAF-1; 4-1BB Saoulli, etal. J Exp Med 1998, 187: 1849-1862, Arch, et al. Mol Cell Biol 1998, 18:558-565), TRAF-2 (4-1BB and OX40 ligand; Saoulli, et al. J Exp Med 1998,187: 1849-1862; Oshima, et al. Int Immunol 1998, 10: 517-526, Kawamata,et al. J Biol Chem 1998, 273: 5808-5814), TRAF-3 (4-1BB and OX40 ligand;Arch, et al. Mol Cell Biol 1998, 18: 558-565; Jang, et al. BiochemBiophys Res Commun 1998, 242: 613-620; Kawamata S, et al., J Biol Chem1998, 273: 5808-5814), OX40L (OX40 ligand; Gramaglia, et al. J Immunol1998, 161: 6510-6517), TRAF-5 (OX40 ligand; Arch, et al. Mol Cell Biol1998, 18: 558-565; Kawamata, et al. J Biol Chem 1998, 273: 5808-5814),and CD70 (CD27 ligand; Couderc, et al. Cancer Gene Ther., 5(3): 163-75).CD154 (CD40 ligand or “CD40L”; Gurunathan, et al. J. Immunol., 1998,161: 4563-4571; Sine, et al. Hum. Gene Ther., 2001, 12: 1091-1102) mayalso be suitable.

One or more cytokines may also be suitable co-stimulatory components or“adjuvants”, either as polypeptides or being encoded by nucleic acidscontained within the compositions of the present invention (Parmiani, etal. Immunol Lett 2000 Sep. 15; 74(1): 41-4; Berzofsky, et al. NatureImmunol. 1: 209-219). Suitable cytokines include, for example,interleukin-2 (IL-2) (Rosenberg, et al. Nature Med. 4: 321-327 (1998)),IL-4, IL-7, IL-12 (reviewed by Pardoll, 1992; Harries, et al. J. GeneMed. 2000 July-August; 2(4):243-9; Rao, et al. J. Immunol. 156:3357-3365 (1996)), IL-15 (Xin, et al. Vaccine, 17:858-866, 1999), IL-16(Cruikshank, et al. J. Leuk Biol. 67(6): 757-66, 2000), IL-18 (J. CancerRes. Clin. Oncol. 2001. 127(12): 718-726), GM-CSF (CSF (Disis, et al.Blood, 88: 202-210 (1996)), tumor necrosis factor-alpha (TNF-α), orinterferons such as IFN-α or INF-γ. Other cytokines may also be suitablefor practicing the present invention, as is known in the art.

Chemokines may also be utilized. For example, fusion proteins comprisingCXCL10 (IP-10) and CCL7 (MCP-3) fused to a tumor self-antigen have beenshown to induce anti-tumor immunity (Biragyn, et al. Nature Biotech.1999, 17: 253-258). The chemokines CCL3 (MIP-1α) and CCL5 (RANTES)(Boyer, et al. Vaccine, 1999, 17 (Supp. 2): S53-S64) may also be of usein practicing the present invention. Other suitable chemokines are knownin the art.

It is also known in the art that suppressive or negative regulatoryimmune mechanisms may be blocked, resulting in enhanced immuneresponses. For instance, treatment with anti-CTLA-4 (Shrikant, et al.Immunity, 1996, 14: 145-155; Sutmuller, et al. J. Exp. Med., 2001, 194:823-832), anti-CD25 (Sutmuller, supra), anti-CD4 (Matsui, et al. J.Immunol., 1999, 163: 184-193), the fusion protein IL13Ra2-Fc (Terabe, etal. Nature Immunol., 2000, 1: 515-520), and combinations thereof (i.e.,anti-CTLA-4 and anti-CD25, Sutmuller, supra) have been shown toupregulate anti-tumor immune responses and would be suitable inpracticing the present invention.

Any of these components may be used alone or in combination with otheragents. For instance, it has been shown that a combination of CD80,ICAM-1 and LFA-3 (“TRICOM”) may potentiate anti-cancer immune responses(Hodge, et al. Cancer Res. 59: 5800-5807 (1999). Other effectivecombinations include, for example, IL-12 GM-CSF (Ahlers; et al. J.Immunol., 158: 3947-3958 (1997); Iwasaki, et al. J. Immunol. 158:4591-4601 (1997)), IL-12+GM-CSF+TNF-α (Ahlers, et al. Int. Immunol. 13:897-908 (2001)), CD80+IL-12 (Fruend, et al. Int. J. Cancer, 85: 508-517(2000); Rao, et al. supra), and CD86+GM-CSF+IL-12 (Iwasaki, supra). Oneof skill in the art would be aware of additional combinations useful incarrying out the present invention. In addition, the skilled artisanwould be aware of additional reagents or methods that may be used tomodulate such mechanisms. These reagents and methods, as well as othersknown by those of skill in the art, may be utilized in practicing thepresent invention.

Additional strategies for improving the efficiency of nucleic acid-basedimmunization may also be used including, for example, the use ofself-replicating viral replicons (Caley, et al. b 1999 . Vaccine, 17:3124-2135; Dubensky, et al. 2000. Mol. Med. 6: 723-732; Leitner, et al.2000. Cancer Res. 60: 51-55), codon optimization (Liu, et al. 2000. Mol.Ther., 1: 497-500; Dubensky, supra; Huang, et al. 2001. J. Virol. 75:4947-4951), in vivo electroporation (Widera, et al. 2000. J. Immunol.164: 4635-3640), incorporation of CpG stimulatory motifs (Gurunathan, etal. Ann. Rev. Immunol., 2000, 18: 927-974; Leitner, supra; Cho, et al.J. Immunol. 168(10):4907-13), sequences for targeting of the endocyticor ubiquitin-processing pathways (Thomson, et al. 1998. J. Virol 72:2246-2252; Velders, et al. 2001. J. Immunol. 166: 5366-5373), Marek'sdisease virus type 1 VP22 sequences (J. Virol. 76(6):2676-82, 2002),prime-boost regimens (Gurunathan, supra; Sullivan, et al. 2000. Nature,408: 605-609; Hanke, et al. 1998. Vaccine, 16: 439-445; Amara, et al.2001. Science, 292: 69-74), and the use of mucosal delivery vectors suchas Salmonella (Darji, et al. 1997. Cell, 91: 765-775; Woo, et al. 2001.Vaccine, 19: 2945-2954). Other methods are known in the art, some ofwhich are described below.

Chemotherapeutic agents, radiation, anti-angiogenic compounds, or otheragents may also be utilized in treating and/or preventing cancer usingimmunogenic targets (Sebti, et al. Oncogene 2000 Dec. 27;19(56):6566-73). For example, in treating metastatic breast cancer,useful chemotherapeutic agents include cyclophosphamide, doxorubicin,paclitaxel, docetaxel, navelbine, capecitabine, and mitomycin C, amongothers. Combination chemotherapeutic regimens have also proven effectiveincluding cyclophosphamide+methotrexate+5-fluorouracil;cyclophosphamide+doxorubicin+5-fluorouracil; or,cyclophosphamide+doxorubicin, for example. Other compounds such asprednisone, a taxane, navelbine, mitomycin C, or vinblastine have beenutilized for various reasons. A majority of breast cancer patients haveestrogen-receptor positive (ER+) tumors and in these patients, endocrinetherapy (i.e., tamoxifen) is preferred over chemotherapy. For suchpatients, tamoxifen or, as a second line therapy, progestins(medroxyprogesterone acetate or megestrol acetate) are preferred.Aromatase inhibitors (i.e., aminoglutethimide and analogs thereof suchas letrozole) decrease the availability of estrogen needed to maintaintumor growth and may be used as second or third line endocrine therapyin certain patients.

Other cancers may require different chemotherapeutic regimens. Forexample, metastatic colorectal cancer is typically treated withCamptosar (irinotecan or CPT-11), 5-fluorouracil or leucovorin, alone orin combination with one another. Proteinase and integrin inhibitors suchas as the MMP inhibitors marimastate (British Biotech), COL-3(Collagenex), Neovastat (Aeterna), AG3340 (Agouron), BMS-275291 (BristolMyers Squibb), CGS 27023A (Novartis) or the integrin inhibitors Vitaxin(Medimmune), or MED1522 (Merck KgaA) may also be suitable for use. Assuch, immunological targeting of immunogenic targets associated withcolorectal cancer could be performed in combination with a treatmentusing those chemotherapeutic agents. Similarly, chemotherapeutic agentsused to treat other types of cancers are well-known, in the art and maybe combined with the immunogenic targets described herein.

Many anti-angiogenic agents are known in the art and would be suitablefor co-administration with the immunogenic target vaccines (see, forexample, Timar, et al. 2001. Pathology Oncol. Res., 7(2): 85-94). Suchagents include, for example, physiological agents such as growth factors(i.e., ANG-2, NK1,2,4 (HOF), transforming growth factor beta (TGF-β)),cytokines (i.e., interferons such as IFN-α, -β, -γ, platelet factor 4(PF-4), PR-39), proteases (i.e., cleaved AT-III, collagen XVIII fragment(Endostatin)), HmwKallikrein-d5 plasmin fragment (Angiostatin),prothrombin-F1-2, TSP-1), protease inhibitors (i.e., tissue inhibitor ofmetalloproteases such as TIMP-1, -2, or -3; maspin; plasminogenactivator-inhibitors such as PAI-1; pigment epithelium derived factor(PEDF)), Tumstatin (available through ILEX, Inc.), antibody products(i.e., the collagen-binding antibodies HUIV26, HUI77, XL313; anti-VEGF;anti-integrin (i.e., Vitaxin, (Lxsys))), and glycosidases (i.e.,heparinase-I, -III). “Chemical” or modified physiological agents knownor believed to have anti-angiogenic potential include, for example,vinblastine, taxol, ketoconazole, thalidomide, dolestatin, combrestatinA, rapamycin (Guba, et al. 2002, Nature Med., 8: 128-135), CEP-7055(available from Cephalon, Inc.), flavone acetic acid, Bay 12-9566 (BayerCorp.), AG3340 (Agouron, Inc.), CGS 27023A (Novartis), tetracylcinederivatives (i.e., COL-3 (Collagenix, Inc.)), Neovastat (Aeterna),BMS-275291 (Bristol-Myers Squibb), low dose 5-FU, low dose methotrexate(MTX), irsofladine, radicicol, cyclosporine, captopril, celecoxib,D45152-sulphated polysaccharide, cationic protein (Protamine), cationicpeptide-VEGF, Suramin (polysulphonated napthyl urea), compounds thatinterfere with the function or production of VEGF (i.e., SU5416 orSU6668 (Sugen), PTK787/ZK22584 (Novartis)), Distamycin A, Angiozyme(ribozyme), isoflavinoids, staurosporine derivatives, genistein,EMD121974 (Merck KcgaA), tyrphostins, isoquinolones, retinoic acid,carboxyamidotriazole, TNP-470, octreotide, 2-methoxyestradiol,aminosterols (i.e., squalamine), glutathione analogues (i.e.,N-acteyl-L-cysteine), combretastatin A-4 (Oxigene), Eph receptorblocking agents (Nature, 414:933-938, 2001), Rh-Angiostatin,Rh-Endostatin (WO 01/93897), cyclic-RGD peptide, accutin-disintegrin,benzodiazepenes, humanized anti-avb3 Ab, Rh-PAI-2, amiloride,p-amidobenzamidine, anti-uPA ab, anti-uPAR Ab,L-phanylalanin-N-methylamides (i.e., Batimistat, Marimastat), AG3340,and minocycline. Many other suitable agents are known in the art andwould suffice in practicing the present invention.

The present invention may also be utilized in combination with“non-traditional” methods of treating cancer. For example, it hasrecently been demonstrated that administration of certain anaerobicbacteria may assist in slowing tumor growth. In one study, Clostridiumnovyi was modified to eliminate a toxin gene carried on a phage episomeand administered to mice with colorectal tumors (Dang, et al. P.N.A.S.USA, 98(26): 15155-15160, 2001). In combination with chemotherapy, thetreatment was shown to cause tumor necrosis in the animals. The reagentsand methodologies described in this application may be combined withsuch treatment methodologies.

Nucleic acids encoding immunogenic targets may be administered topatients by any of several available techniques. Various viral vectorsthat have been successfully utilized for introducing a nucleic acid to ahost include retrovirus, adenovirus, adeno-associated virus (AAV),herpes virus, and poxvirus, among others. It is understood in the artthat many such viral vectors are available in the art. The vectors ofthe present invention may be constructed using standard recombinanttechniques widely available to one skilled in the art. Such techniquesmay be found in common molecular biology references such as MolecularCloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring HarborLaboratory Press), Gene Expression Technology (Methods in Enzymology,Vol. 185, edited by D. Goeddel, 1991. Academic Press, San Diego,Calif.), and PCR Protocols: A Guide to Methods and Applications (Innis,et al. 1990. Academic Press, San Diego, Calif.).

Preferred retroviral vectors are derivatives of lentivirus as well asderivatives of murine or avian retroviruses. Examples of suitableretroviral vectors include, for example, Moloney murine leukemia virus(MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumorvirus (MuMTV), SIV, BJV, HIV and Rous Sarcoma Virus (RSV). A number ofretroviral vectors can incorporate multiple exogenous nucleic acidsequences; As recombinant retroviruses are defective, they requireassistance in order to produce infectious vector particles. Thisassistance can be provided by, for example, helper cell lines encodingretrovirus structural genes. Suitable helper cell lines include ψ2,PA317 and PA12, among others. The vector virions produced using suchcell lines may then be used to infect a tissue cell line, such as NIH3T3 cells, to produce large quantities of chimeric retroviral virions.Retroviral vectors may be administered by traditional methods (i.e.,injection) or by implantation of a “producer cell line” in proximity tothe target cell population (Culver, K., et al., 1994, Hum. Gene Ther., 5(3): 343-79; Culver, K., et al., Cold Spring Harb. Symp. Quant. Biol.,59: 685-90); Oldfield, E., 1993, Hum. Gene Ther., 4 (1): 39-69). Theproducer cell line is engineered to produce a viral vector and releasesviral particles in the vicinity of the target cell. A portion of thereleased viral particles contact the target cells and infect thosecells, thus delivering a nucleic acid of the present invention to thetarget cell. Following infection of the target cell, expression of thenucleic acid of the vector occurs.

Adenoviral vectors have proven especially useful for gene transfer intoeukaryotic cells (Rosenfeld, M., et al., 1991, Science, 252 (5004):431-4; Crystal, R., et al., 1994, Nat. Genet., 8 (I): 42-51), the studyeukaryotic gene expression (Levrero, M., et al., 1991, Gene, 101 (2):195-202), vaccine development (Graham, F. and Prevec, L., 1992,Biotechnology, 20: 363-90), and in animal models (Stratford-Perricaudet,L., et al., 1992, Bone Marrow Transplant., 9 (Suppl. 1): 151-2; Rich,D., et al., 1993, Hum. Gene Ther., 4 (4): 461-76). Experimental routesfor administrating recombinant Ad to different tissues in vivo haveincluded intratracheal instillation (Rosenfeld, M., et al., 1992, Cell,68 (1): 143-55) injection into muscle (Quentin, B., et al., 1992, Proc.Natl. Acad. Sci. U.S.A., 89 (7): 2581-4), peripheral intravenousinjection (Herz, J., and Gerard, R., 1993, Proc. Natl. Acad. Sci.U.S.A., 90 (7): 2812-6) and stereotactic inoculation to brain (Le Gal LaSalle, G., et al., 1993, Science, 259 (5097): 988-90), among others.

Adeno-associated virus (AAV) demonstrates high-level infectivity, broadhost range and specificity in integrating into the host cell genome(Hermonat, P., et al., 1984, Proc. Natl. Acad. Sci. U.S.A., 81 (20):6466-70). And Herpes Simplex Virus type-1 (HSV-1) is yet anotherattractive vector system, especially for use in the nervous systembecause of its neurotropic property (Geller, A., et al., 1991, TrendsNeurosci., 14 (10): 428-32; Glorioso, et al., 1995, Mol. Biotechnol., 4(1): 87-99; Glorioso, et al., 1995, Annu. Rev. Microbiol., 49: 675-710).

Poxvirus is another useful expression vector (Smith, et al. 1983, Gene,25 (1): 21-8; Moss, et al, 1992, Biotechnology, 20: 345-62; Moss, et al,1992, Curr. Top: Microbiol. Immunol., 158: 25-38; Moss, et al. 1991.Science, 252: 1662-1667). Poxviruses shown to be useful includevaccinia, NYVAC, avipox, fowlpox, canarypox, ALVAC, and ALVAC(2), amongothers.

NYVAC (vP866) was derived from the Copenhagen vaccine strain of vacciniavirus by deleting six nonessential regions of the genome encoding knownor potential virulence factors (see, for example, U.S. Pat. Nos.5,364,773 and 5,494,807). The deletion loci were also engineered asrecipient loci for the insertion of foreign genes. The deleted regionsare: thymidine kinase gene (TK; J2R); hemorrhagic region (u; B13R+B14R);A type inclusion body region (ATI; A26L); hemagglutinin gene (HA; A56R);host range gene region (C7L-K1L); and, large subunit, ribonucleotidereductase (I4L). NYVAC is a genetically engineered vaccinia virus strainthat was generated by the specific deletion of eighteen open readingframes encoding gene products associated with virulence and host range.NYVAC has been show to be useful for expressing TAs (see, for example,U.S. Pat. No. 6,265,189). NYVAC (vP866), vP994, vCP205, vCP1433,placZH6H4Lreverse, pMPC6H6K3E3 and pC3H6FHVB were also deposited withthe ATCC under the terms of the Budapest Treaty, accession numbersVR-2559, VR-2558, VR-2557, VR-2556, ATCC-97913, ATCC-97912, andATCC-97914, respectively.

ALVAC-based recombinant viruses (i.e., ALVAC-1 and ALVAC-2) are alsosuitable for use in practicing the present invention (see, for example.U.S. Pat. No. 5,756,103). ALVAC(2) is identical to ALVAC(1) except thatALVAC(2) genome comprises the vaccinia E3L and K3L genes under thecontrol of vaccinia promoters (U.S. Pat. No. 6,130,066; Beattie et al.,1995a, 1995b, 1991; Chang et al., 1992; Davies et al., 1993). BothALVAC(1) and ALVAC(2) have been demonstrated to be useful in expressingforeign DNA sequences, such as TAs (Tartaglia et to al., 1993 a,b; U.S.Pat. No. 5,833,975). ALVAC was deposited under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110-2209, USA, ATCC accessionnumber VR-2547.

Another useful poxvirus vector is TROVAC. TROVAC refers to an attenuatedfowlpox that was a plaque-cloned isolate derived from the FP-1 vaccinestrain of fowlpoxvirus which is licensed for vaccination of 1 day oldchicks. TROVAC was likewise deposited under the terms of the BudapestTreaty with the ATCC, accession number 2553.

“Non-viral” plasmid vectors may also be suitable in practicing thepresent invention. Preferred plasmid vectors are compatible withbacterial, insect, and/or mammalian host cells. Such vectors include,for example, PCR-II, pCR3, and pcDNA3.1 (Invitrogen, San Diego, Calif.),pBSII (Stratagene, La Jolla, Calif.), pETI 5 (Novagen, Madison, Wis.),pGEX (Pharmacia Biotech, Piscataway, N.J.), pEGFP-N2 (Clontech, PaloAlto, Calif.), pETL (BlueBacll, Invitrogen), pDSR-alpha (PCT pub. No. WO90/14363) and pFastBacDual (Gibco-BRL, Grand Island, N.Y.) as well asBluescript®) plasmid derivatives (a high copy number COLE I-basedphagemid, Stratagene Cloning Systems, La Jolla, Calif.), PCR cloningplasmids designed for cloning Taq-amplified PCR products (e.g., TOPO™ TACloning® kit, PCR2.1® plasmid derivatives, Invitrogen, Carlsbad,Calif.). Bacterial vectors may also be used with the current invention.These vectors include, for example, Shigella, Salmonella, Vibriocholerae, Lactobacillus, Bacille calmette guerin (BCG), andStreptococcus (see for example, WO 88/6626; WO 90/0594; WO 91/13157; WO92/1796; and WO 92/21376). Many other non-viral plasmid expressionvectors and systems are known in the art and could be used with thecurrent invention.

Suitable nucleic acid delivery techniques include DNA-ligand complexes,adenovirus-ligand-DNA complexes, direct injection of DNA, CaPO₄precipitation, gene gun techniques, electroporation, and colloidaldispersion systems, among others. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome, which are artificial membrane vesicles usefulas delivery vehicles in vitro and in vivo. RNA, DNA and intact virionscan be encapsulated within the aqueous interior and be delivered tocells in a biologically active form (Fraley, R., et al., 1981, TrendsBiochem. Sci., 6: 77). The composition of the liposome is usually acombination of phospholipids, particularlyhigh-phase-transition-temperature phospholipids, usually in combinationwith steroids, especially cholesterol. Other phospholipids or otherlipids may also be used. The physical characteristics of liposomesdepend on pH, ionic strength, and the presence of divalent cations.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Particularly useful arediacylphosphatidylglycerols, where the lipid moiety contains from 14-18carbon atoms, particularly from 16-18 carbon atoms, and is saturated.Illustrative phospholipids include egg phosphatidylcholine,dipalmitoylphosphatidylcholine and distearoylphosphatidylcholine.

An immunogenic target may also be administered in combination with oneor more adjuvants to boost the immune response. Exemplary adjuvants areshown in Table II below:

TABLE II Types of Immunologic Adjuvants Type of Specific Examples/Adjuvant General Examples References Gel-type Aluminum hydroxide/(Aggerbeck and Heron, phosphate (“alum adjuvants”) 1995) Calciumphosphate (Relyveld, 1986) Microbial Muramyl dipeptide (MDP) (Chedid etal., 1986) Bacterial exotoxins Cholera toxin (CT), E. coli labile toxin(LT)(Freytag and Clements, 1999) Endotoxin-based adjuvantsMonophosphoryl lipid A (MPL) (Ulrich and Myers, 1995) Other bacterialCpG oligonucleotides (Corral and Petray, 2000), BCG sequences (Krieg, etal. Nature, 374: 576), tetanus toxoid (Rice, et al. J. Immunol., 2001,167: 1558-1565) Particulate Biodegradable (Gupta et al., 1998) Polymermicrospheres Immunostimulatory (Morein and Bengtsson, complexes (ISCOMs)1999) Liposomes (Wassef et al., 1994) Oil-emulsion Freund's incompleteadjuvant (Jensen et al., 1998) and Microfluidized emulsions MF59 (Ott etal., 1995) surfactant- SAF (Allison and Byars, based 1992) (Allison,1999) adjuvants Saponins QS-21 (Kensil, 1996) Synthetic Muramyl peptidederivatives Murabutide (Lederer, 1986) Threony-MDP (Allison, 1997)Nonionic block copolymers L121 (Allison, 1999) Polyphosphazene (PCPP)(Payne et al., 1995) Synthetic polynucleotides Poly A:U. Poly I:C(Johnson, 1994) Thalidomide derivatives CC-4047/ACTIMID (J. Immunol.,168(10): 4914-9)

The immunogenic targets of the present invention may also be used togenerate antibodies for use in screening assays or for immunotherapy,which are another aspect of the present invention. Other uses would beapparent to one of skill in the art. The term “antibody” includesantibody fragments, as are known in the art, including Fab, Fab₂, singlechain antibodies (Fv for example), humanized antibodies, chimericantibodies, human antibodies, produced by several methods as are knownin the art.

Methods of preparing and utilizing various types of antibodies arewell-known to those of skill in the art and would be suitable inpracticing the present invention (see, for example, Harlow, et al.Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988;Harlow, et al. Using Antibodies: A Laboratory Manual, Portable ProtocolNo. 1, 1998; Kohler and Milstein, Nature, 256:495 (1975)); Jones et al.Nature, 321:522-525 (1986); Riechmann et al. Nature, 332:323-329 (1988);Presta (Curr. Op. Struct. Biol., 2:593-596 (1992); Verboeyen et al.(Science, 239:1534-1536 (1988); Hoogenboom et al., J. Mol. Biol.,227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991); Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985); Boerner et al., J. Immunol., 147(1):86-95 (1991); Marks et al.,Bio/Technology 10, 779-783 (1992); Lonberg et al., Nature 368 856-859(1994); Morrison, Nature 368 812-13 (1994); Fishwild et al., NatureBiotechnology 14, 845-51 (1996); Neuberger, Nature Biotechnology 14, 826(1996); Lonberg and Huszar, Intern. Rev. Immunol. 13 65-93 (1995); aswell as U.S. Pat. Nos. 4,816,567; 5,545,807; 5,545,806; 5,569,825;5,625,126; 5,633,425; and, 5,661,016). The antibodies or derivativestherefrom may also be conjugated to therapeutic moieties such ascytotoxic drugs or toxins, or active fragments thereof such as diptheriaA chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin,phenomycin, enomycin, among others. Cytotoxic agents may also includeradiochemicals. Antibodies and their derivatives may be incorporatedinto compositions of the invention for use in vitro or in vivo.

Nucleic acids, proteins, or derivatives thereof representing animmunogenic target may be used in assays to determine the presence of adisease state in a patient, to predict prognosis, or to determine theeffectiveness of a chemotherapeutic or other treatment regimen.Expression profiles, performed as is known in the art, may be used todetermine the relative level of expression of the immunogenic target.The level of expression may then be correlated with base levels todetermine whether a particular disease is present within the patient,the patient is prognosis, or whether a particular treatment regimen iseffective. For example, if the patient is being treated with aparticular chemotherapeutic regimen, a decreased level of expression ofan immunogenic target in the patient's tissues (i.e., in peripheralblood) may indicate the regimen is decreasing the cancer load in thathost. Similarly, if the level of expression is increasing, anothertherapeutic modality may need to be utilized. In one embodiment, nucleicacid probes corresponding to a nucleic acid encoding an immunogenictarget may be attached to a biochip, as is known in the art, for thedetection and quantification of expression in the host.

It is also possible to use nucleic acids, proteins, derivativestherefrom, or antibodies thereto as reagents in drug screening assays.The reagents may be used to ascertain the effect of a drug candidate onthe expression of the immunogenic target in a cell line, or a cell ortissue of a patient. The expression profiling technique may be combinedwith high throughput screening techniques to allow rapid identificationof useful compounds and monitor the effectiveness of treatment with adrug candidate (see, for example, Zlokarnik, et al., Science 279, 84-8(1998)). Drug candidates may be chemical compounds, nucleic acids,proteins, antibodies, or derivatives therefrom, whether naturallyoccurring or synthetically derived. Drug candidates thus identified maybe utilized, among other uses, as pharmaceutical compositions foradministration to patients or for use in further screening assays.

Administration of a composition of the present invention to a host maybe accomplished using any of a variety of techniques known to those ofskill in the art. The composition(s) may be processed in accordance withconventional methods of pharmacy to produce medicinal agents foradministration to patients, including humans and other mammals (i.e., a“pharmaceutical composition”). The pharmaceutical composition ispreferably made in the form of a dosage unit containing a given amountof DNA, viral vector particles, polypeptide or peptide, for example. Asuitable daily dose for a human or other mammal may vary widelydepending on the condition of the patient and other factors, but, onceagain, can be determined using routine methods.

The pharmaceutical composition may be administered orally, parentally,by inhalation spray, rectally, intranodally, or topically in dosage unitformulations containing conventional pharmaceutically acceptablecarriers, adjuvants, and vehicles. The term “pharmaceutically acceptablecarrier” or “physiologically acceptable carrier” as used herein refersto one or more formulation materials suitable for accomplishing orenhancing the delivery of a nucleic acid, polypeptide, or peptide as apharmaceutical composition. A “pharmaceutical composition” is acomposition comprising a therapeutically effective amount of a nucleicacid or polypeptide. The terms “effective amount” and “therapeuticallyeffective amount” each refer to the amount of a nucleic acid orpolypeptide used to induce or enhance an effective immune response. Itis preferred that compositions of the present invention provide for theinduction or enhancement of an anti-tumor immune response in a hostwhich protects the host from the development of a tumor and/or allowsthe host to eliminate an existing tumor from the body.

For oral administration, the pharmaceutical composition may be of any ofseveral forms including, for example, a capsule, a tablet, a suspension,or liquid, among others. Liquids may be administered by injection as acomposition with suitable carriers including saline, dextrose, or water.The term parenteral as used herein includes subcutaneous, intravenous,intramuscular, intrasternal, infusion, or intraperitonealadministration. Suppositories for rectal administration of the drug canbe prepared by mixing the drug with a suitable non-irritating excipientsuch as cocoa butter and polyethylene glycols that are solid at ordinarytemperatures but liquid at the rectal temperature.

The dosage regimen for immunizing a host or otherwise treating adisorder or a disease with a composition of this invention is based on avariety of factors, including the type of disease, the age, weight, sex,medical condition of the patient, the severity of the condition, theroute of administration, and the particular compound employed. Forexample, a poxyiral vector may be administered as a compositioncomprising 1×10⁶ infectious particles per dose. Thus, the dosage regimenmay vary widely, but can be determined routinely using standard methods.

A prime-boost regimen may also be utilized (see, for example, WO01/30382 A1) in which the targeted immunogen is initially administeredin a priming step in one form followed by a boosting step in which thetargeted immunogen is administered in another form. The form of thetargeted immunogen in the priming and boosting steps are different. Forinstance, if the priming step utilized a nucleic acid, the boost may beadministered as a peptide. Similarly, where a priming step utilized onetype of recombinant virus (i.e., ALVAC), the boost step may utilizeanother type of virus (i.e., NYVAC). This prime-boost method ofadministration has been shown to induce strong immunological responses.

While the compositions of the invention can be administered as the soleactive pharmaceutical agent, they can also be used in combination withone or more other compositions or agents (i.e., other immunogenictargets, co-stimulatory molecules, adjuvants). When administered as acombination, the individual components can be formulated as separatecompositions administered at the same time or different times, or thecomponents can be combined as a single composition.

Injectable preparations, such as sterile injectable aqueous oroleaginous suspensions, may be formulated according to known methodsusing suitable dispersing or wetting agents and suspending agents. Theinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent.Suitable vehicles and solvents that may be employed are water, Ringer'ssolution, and isotonic sodium chloride solution, among others. Forinstance, a viral vector such as a poxvirus may be prepared in 0.4% toNaCl. In addition, sterile, fixed oils are conventionally employed as asolvent or suspending medium. For this purpose, any bland fixed oil maybe employed, including synthetic mono- or diglycerides. In addition,fatty acids such as oleic acid find use in the preparation ofinjectables.

For topical administration, a suitable topical dose of a composition maybe administered one to four, and preferably two or three times daily.The dose may also be administered with intervening days during which nodoes is applied. Suitable compositions may comprise from 0.001% to 10%w/w, for example, from 1% to 2% by weight of the formulation, althoughit may comprise as much as 10% w/w, but preferably not more than 5% w/w,and more preferably from 0.1% to 1% of the formulation. Formulationssuitable for topical administration include liquid or semi-liquidpreparations suitable for penetration through the skin (e.g., liniments,lotions, ointments, creams, or pastes) and drops suitable foradministration to the eye, ear, or nose.

The pharmaceutical compositions may also be prepared in a solid form(including granules, powders or suppositories). The pharmaceuticalcompositions may be subjected to conventional pharmaceutical operationssuch as sterilization and/or may contain conventional adjuvants, such aspreservatives, stabilizers, wetting agents, emulsifiers, buffers etc.Solid dosage forms for oral administration may include capsules,tablets, pills, powders, and granules. In such solid dosage forms, theactive compound may be admixed with at least one inert diluent such assucrose, lactose, or starch. Such dosage forms may also comprise, as innormal practice, additional substances other than inert diluents, e.g.,lubricating agents such as magnesium stearate. In the case of capsules,tablets, and pills, the dosage forms may also comprise buffering agents.Tablets and pills can additionally be prepared with enteric coatings.Liquid dosage forms for oral administration may include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirscontaining inert diluents commonly used in the art, such as water. Suchcompositions may also comprise adjuvants, such as wetting sweetening,flavoring, and perfuming agents.

Pharmaceutical compositions comprising a nucleic acid or polypeptide ofthe present invention may take any of several forms and may beadministered by any of several routes. In preferred embodiments, thecompositions are administered via a parenteral route (intradermal,intramuscular or subcutaneous) to induce an immune response in the host.Alternatively, the composition may be administered directly into a lymphnode (intranodal) or tumor mass (i.e., intratumoral administration). Forexample, the dose could be administered subcutaneously at days 0, 7, and14. Suitable methods for immunization using compositions comprising TAsare known in the art, as shown for p53 (Hollstein et al., 1991), p21-ras(Almoguera et al., 1988), HER-2 (Fendly et al., 1990), themelanoma-associated antigens (MAGE-1; MAGE-2) (van der Bruggen et al.,1991), p97 (Hu et al., 1988), melanoma-associated antigen E (WO99/30737) and carcinoembryonic antigen (CEA) (Kantor et al., 1993;Fishbein et al., 1992; Kaufman et al., 1991), among others.

Preferred embodiments of administratable compositions include, forexample, nucleic acids or polypeptides in liquid preparations such assuspensions, syrups, or elixirs. Preferred injectable preparationsinclude, for example, nucleic acids or polypeptides suitable forparental, subcutaneous, intradermal, intramuscular or intravenousadministration such as sterile suspensions or emulsions. For example, arecombinant poxvirus may be in admixture with a suitable carrier,diluent, or excipient such as sterile water, physiological saline,glucose or the like. The composition may also be provided in lyophilizedform for reconstituting, for instance, in isotonic aqueous, salinebuffer. In addition, the compositions can be co-administered orsequentially administered with other antineoplastic, anti-tumor oranti-cancer agents and/or with agents which reduce or alleviate illeffects of antineoplastic, anti-tumor or anti-cancer agents.

A kit comprising a composition of the present invention is alsoprovided. The kit can include a separate container containing a suitablecarrier, diluent or excipient. The kit can also include an additionalanti-cancer, anti-tumor or antineoplastic agent and/or an agent thatreduces or alleviates ill effects of antineoplastic, anti-tumor oranti-cancer agents for co- or sequential-administration. Additionally,the kit can include instructions for mixing or combining ingredientsand/or administration.

A better understanding of the present invention and of its manyadvantages will be had from the following examples, given by way ofillustration.

EXAMPLES Example 1 BFA5 Breast Cancer Antigen A. Identification of BFA5

Microarray profiling analysis indicated that BFA5 was expressed at lowto high levels in 41 out of 54 breast tumor biopsy samples (76%) and athigh levels in 31 out of 54 breast tumors (57%), as compared to a panelof 52 normal, non-tumor tissues. In situ hybridization (ISH) wasperformed using a series of BFA5 DNA probes and confirmed the microarraywith at least 61% of the tumors showing fairly strong signals. Furtherbioinformatics assessment confirmed the results of these gene expressionanalysis results.

Sequence analysis of the BFA5 nucleotide sequence revealed a high degreeof similarity to two unidentified human genes: KIAA1074 (GenBankAccession No. XM_(—)159732); and, KIAA0565 (GenBank Accession No.AB011137) isolated from a number of fetal and adult brain cDNA clones(Kikuno, et al. The complete sequences of 100 new cDNA clones from brainwhich code for large proteins in vitro. DNA Res. 6: 197-205). Thesegenes were found to contain putative Zn finger regions and a nuclearlocalization sequence. BFA5 was suggested by others to be a potentialbreast cancer antigen (Jager, et al. 2001. Identification of atissue-specific putative transcription factor in breast tissue byserological screening of a breast cancer library. Cancer Res. 61:2055-2061 and WO 01/47959). In each of these publications, thenucleotide sequence BFA5 was designated NYBR-1 (“New York BreastCancer-1”; GenBank Accession Nos. AF269087 (nucleotide) and AAK27325(amino acid).

As shown previously by Jager, et al. and described in WO 01/47959,supra, BFA5 is specifically expressed in mammary gland, being expressedin 12/19 breast tumors analyzed. The structure of the BFA5/NYBR-1 genehas revealed that it encodes a 150-160 kD nuclear transcription factorwith a bZIP site (DNA-binding domain followed by a leucine zippermotif). The gene also contains 5 tandem ankyrin repeats implying a rolein protein-protein interactions. These ankyrin repeats may play a rolein homo-dimerization of the protein. The BFA5 cDNA sequence is shown inFIG. 4 and SEQ ID NO.: S. The BFA5 amino acid sequence is shown in FIG.5 and SEQ ID NO.: 6.

B. Immunoreactivity of BFA5 1. Activation of Human T Cells and IFN-γSecretion in ELISPOT.

A library of 100 peptides from the BFA5/NYBR-1 coding sequence that arepredicted to be medium to high binders to HLA-A*0201 were designed usingRammensee and Parker algorithms. The library was sub-divided into 10pools of ten peptides (Table III), and each pool was used to activate 10different T cell cultures after pulsing peptides on to mature autologousdendritic cells. Two experiments were performed with the library ofBFA5/NYBR-1 peptides demonstrating immunoreactivity in HLA-A*0201 humanT cells, as described below.

TABLE III BFA5 Peptide Pools Peptide Group CLP number Sequence BFA5 2983LMOMQTFKA Group 1 2984 KVSIPTKAL 2985 SIPTKALEL 2986 LELKNEQTL 2987TVSQKDVCL 2988 SVPNKALEL 2989 CETVSQKDV 2990 KINGKLEES 2991 SLVEKTPDE2992 SLCETVSQK BFA5 2993 ESDKINGKL Group 2 2994 MLLQQNVDV 2995 NMWLQQQLV2996 FLVDRKCQL 2997 YLLHENCML 2998 SLFESSAKI 2999 KITIDIHFL 3000QLQSKNMWL 3001 SLDQKLFQL 3002 FLLIKNANA BFA5 3003 KILDTVHSC Group 3 3004SLSKILDTV 3005 ILIDSGADI 3006 KVMEINREV 3007 KLLSHGAVI 3009 AVYSEILSV3010 KMNVDVSST 3011 ILSVVAKLL 3012 VLIAENTML BFA5 3013 KLSKNHQNT Group 43014 SLTPLLLSI 3015 SQYSGQLKV 3016 KELEVKQQL 3017 QIMEYIRKL 3018AMLKLEIAT 3019 VLHQPLSEA 3020 GLLKATCGM 3021 GLLKATCGM 3022 QQLEQALRIBFA5 3023 CMLKKEIAM Group 5 3024 EQMKKKFCV 3025 IQDIELKSV 3026 SVPNKAFEL3027 SIYQKVMEI 3028 NLNYAGDAL 3029 AVQDHDQIV BFA5 3033 FESSAKIOV Group 63034 GVTAEHYAV 3035 RVTSNKTKV 3036 TVSQKDVCV 3037 KSOEPAPHI 3038KVLIAENTM 3039 MLKLEIATL 3040 EILSVVAKL 3041 MLKKEIAML 3042 LLKEKNEEIBFA5 3043 ALRIQDIEL Group 7 3044 KIREELGRI 3045 TLKLKEESL 3046 ILNEKIREE3047 VLKKKLSEA 3048 GTSOKIQCL 3049 GADINLVDV 3050 ELCSVRLTL 3051SVESNLNQV 3052 SLKINLNYA BFA5 3053 KTPOEAASL Group 8 3054 ATCGMKVSI 3055LSHGAVIEV 3056 EIAMLKLEI 3057 AELQMTLKL 3058 VFAADICGV 3060 PAIEMQNSV3061 EIFNYNNHL 3062 ILKERMAEL BFA5 3063 QLVHAHKKA Group 9 3065 NIQDAQKRT3066 NLVDVYGNM 3067 KCTALMLAV 3068 KIQCLEKAT 3069 KIAWEKKET 3070IAWEKKEDT 3071 VGMLLQQNV 3072 VKTGCVARV BFA5 3074 ALHYAVYSE Group 103075 QMKKKFCVL 3076 ALQCHQEAC 3077 SEQIVEFLL 3078 AVIEVHNKA 3079AVTCGFHHI 3080 ACLQRKMNV 3081 SLVEGTSDK

ELISPOT analysis was performed on human T-cell cultures activatedthrough four rounds of stimulation with each pool of BFA5 peptides.Reactivity against a CMV pp65 peptide and a Flu matrix peptide were usedas positive controls for T-cell activation in the experiments. Eachexperiment was performed with PBMC and dendritic cells from a singleHLA-A*0201⁺ donor designated as “AP10”. The results show that, althoughBFA4 is markedly reactive with high ELISPOT counts per 100,000 cells inthe assay, BFA5 is even more reactive with 9/10 pools demonstratingELISPOT reactivity. Similar results were obtained for both BFA4 andBFA5/NYBR-1 with a different HLA-A*0201. The bars reach a maximum at 600spots because beyond that the ELISPOT reader does not give accuratecounts. Cultures having a reading of 600 spots have more than thisnumber of spots.

A large number of the BFA5 peptide pools of are reactive as shown by thehigh levels of IFN-γ production. Each reactive peptide pool was thenseparated into individual peptides and analyzed for immunogencity usingELISPOT analysis to isolate single reactive BFA5 peptides. BFA5 ishighly immunogenic with several reactive single peptides than that ofBFA4. Similar results were obtained in two independent PBMC cultureexperiments.

In addition to ELISPOT analysis, human T cells activated by BFA5peptides were assayed to determine their ability to function as CTL. Thecells were activated using peptide-pulsed dendritic cells followed byCD40 ligand-activated B cells (5 rounds of stimulation). The experimentshown was performed with isolated PBMC from HLA-A*0201⁺ donor AP31.Isolated T cells were tested in ⁵¹Cr-release assays using peptide-loadedT2 cells. The % specific lysis at a 10:1, 5:1, and 1:1 T-cell to targetratio is shown for T2 cells pulsed with either pools of BFA5/NYBR-1peptides or with individual peptide's. The graph shows CTL activityinduced against targets loaded with a c non-specific HLA-A*0201-bindingHIV peptide (control) followed by the CTL activity against the peptidepool (Pool 1 etc.) and then the activity induced by individual peptidesfrom the respective pool to the right. A high level of cytotoxicity wasobserved for some peptides at a 1:1 E:T ratio. CTL activity (percentspecific lysis) induced by the control HTV peptide was generally <10%.Similar results were obtained with another PBMC donor expressingHLA-A*0201 (AP10). A large number of BFA5 peptides trigger Tcell-mediated cytotoxicity of BFA5 peptide-loaded target cells. Table IVlists those peptides having immunogenic properties. Five peptides(LMDMQTFKA, ILIDSGADI, 1LSVVAICLL, SQYSGQLKV, and ELCSVRLTL) were foundto induce both IFN-γ secretion and CTL activity in T cells from bothdonors.

TABLE IV Immunoreactive peptides from BFA5 BFA5 peptides elicitingBFA5 peptides high IFN-γ release inducing CTL lysis(>200 spots/100,000 cells) of pulsed cells Donor AP10 Donor AP31Donor AP10 Donor AP31 LMDMQTFKA LMDMQTFKA LMDMQTFKA LMOMQTFKA KVSIPTKALKVSIPTKAL SIPTKALEL SIPTKALEL TVSQKDVCL SVPNKALEL YLLHENCML YLLHENCMLYLLHENCML QLQSKNMWL QLQSKNMWL QLQSKNMWL SLSKILDTV SLSKILDTV SLSKILDTVILIDSGADI ILIDSGADI ILIDSGADI ILIDSGADI KVMEINREV AVYSEILSV ILSVVAKLLILSVVAKLL ILSVVAKLL ILSVVAKLL SLTPLLLSI SLTPLLLSI SLTPLLLSI SQYSGQLKVSQYSGQLKV SQYSGQLKV SQYSGQLKV QIMEYIRKL QIMEYIRKL QIMEYIRKL SVPNKAFELNLNYAGDAL NLNYAGDAL GVTAEHYAV KSQEPAFHI MLKLEIATL MLKLEIATL MLKLEIATLMLKKEIAML ALRIQDIEL VLKKKLSEA ELCSVRLTL ELCSVRLTL ELCSVRLTL ELCSVRLTLSLKINLNYA SLKINLNYA SLKINLNYA ATCGMKVSI ATCGMKVSI AELQMTLKL AELQMTLKLAELQMTLKL VFAADICGV ILKEKNAEL ILKEKNAEL NLVDVYGNM NLVDVYGNM KCTALMLAV

C. Immunological Reagents

Polyclonal antisera were generated against the following series of 22-to 23-mer peptides of BFA5:

BFA5(1-23) KLH-MTKRKKTINLNIQDAQKRTALHW (CLP-2977) BFA5(312-334)KLH-TSEKFTWPAKGRPRKIAWEKKED (CLP-2978) BFA5(612-634)KLH-DEILPSESKQKDYEENSWDTESL (CLP-2979) BFA5(972-994)KLH-RLTLNQEEEKRRNADILNEKIRE (CLP-2980) BFA5(1117-1139)KLH-AENTMLTSKLKEKQDKEILEAEI (CLP-2981) BFA5(1319-1341)KLH-NYNNHLKNRIYQYEKEKAETENS (CLP-2982)

Prebleed samples from rabbits were processed and stored at −20° C.Rabbits were immunized as follows: 1) the peptides were administered asan emulsion with Freund's Complete Adjuvant (FCA); and, 2) two weekslater, the peptides were coupled with Keyhole-Limpet Hemocyanin(KLH)-coupled and administered as an emulsion with Freund's IncompleteAdjuvant FIA. The following results were observed:

TABLE V IgG titer × 10⁵ (after IgG titer × 10⁵ (after first Immunizationsecond Immunization Peptide/protein Rb1/Rb2) Rb1/Rb2) CLP 2977 25/6 256/64  CLP 2978 25/25  64/256 CLP 2979 12/25 256/512 CLP 2980 25/121024/128  CLP 2981 8/4 256/64  CLP 2982 2/2 64/32Prebleed sample results exhibited IgG titers <100 for all samples.

To assess the quality of the polyclonal antisera, western blots wereperformed using sera against BFA5. Sera were separately screened againstcell extracts obtained from the BT474, MDMB453, MCF-7, Calu-6, and CosA2cells. The approximate expected MW, of BFA5 protein is 153 kDa. A 220 kDband was observed in the BT474 extract with CLP2980 antibody but not inthe MDMB453 cell extracts however a ˜130 kD band was present in theMDMB453 extract. Both bands were found to be consistent with thepolyclonal antibosera tested in this analysis. Neither of these bands ispresent in the negative control. Thus, it can be concluded that thepolyclonal antisera are specific for BFA5.

While the present invention has been described in terms of the preferredembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Therefore, it is intended that theappended claims cover all such equivalent variations that come withinthe scope of the invention as claimed.

1-63. (canceled)
 64. An expression vector comprising the nucleic acidsequence as illustrated in SEQ ID NO.: 5 or a fragment thereof.
 65. Theexpression vector of claim 64 wherein the vector is a plasmid or a viralvector.
 66. The expression vector of claim 65 wherein the viral vectoris selected from the group consisting of poxvirus, adenovirus,retrovirus, herpesvirus, and adeno-associated virus.
 67. The expressionvector of claim 66 wherein the viral vector is a poxvirus selected fromthe group consisting of vaccinia, NYVAC, avipox, canarypox, ALVAC,ALVAC(2), fowlpox, and TROVAC.
 68. The expression vector of claim 66wherein the viral vector is a poxvirus selected from the groupconsisting of NYVAC, ALVAC, and ALVAC(2).
 69. The expression vector ofclaim 1 further comprising at least one additional tumor-associatedantigen.
 70. An isolated peptide derived from BFA5 as shown in Table Xor XI.
 71. A method for immunizing a host against the tumor antigen BFA5comprising administering to the patient a peptide of claim 74, eitheralone or in combination with another agent, where the individualcomponents of the combination are administered simultaneously orseparately from one another.
 72. An expression vector comprising anucleic acid sequence encoding the amino acid sequence of SEQ ID NO.: 6or a fragment thereof.
 73. A composition comprising an expression vectorof claim 1 and a pharmaceutically acceptable carrier.
 74. A compositioncomprising an expression vector of claim 76 and a pharmaceuticallyacceptable carrier.
 75. An antibody having the ability to bind the aminoacid sequence of SEQ ID NO.: 6 or a fragment thereof.
 76. The antibodyof claim 79 wherein the fragment is selected from the group consistingof SEQ ID NO.:7, SEQ ID NO.:8, SEQ ID NO.:9, SEQ ID NO.:11, SEQ IDNO.:12, SEQ ID NO.:21, SEQ ID NO.:24, SEQ ID NO.:29, SEQ ID NO.:30, SEQID NO.:32, SEQ ID NO.:34, SEQ ID NO.:37, SEQ ID NO.:38, SEQ ID NO.:40,SEQ ID NO.:49, SEQ ID NO.:51, SEQ ID NO.:54, SEQ ID NO.:57, SEQ IDNO.:59, SEQ ID NO.:61, SEQ ID NO.:63, SEQ ID NO.:67, SEQ ID NO.:70, SEQID NO.:72, SEQ ID NO.:74, SEQ ID NO.:77, SEQ ID NO.:78, SEQ ID NO.:81,SEQ ID NO.:84, and SEQ ID NO.:85.
 77. A composition comprising anantibody of claim 79 and a pharmaceutically acceptable carrier.
 78. Acomposition comprising an antibody of claim 80 and a pharmaceuticallyacceptable carrier.
 79. An isolated peptide selected from the groupconsisting of SEQ ID NO.:8, SEQ ID NO.:9, SEQ ID NO.:11, and SEQ IDNO.:12.
 80. A composition comprising an isolated peptide of claim 79 anda pharmaceutically acceptable carrier.
 81. A method for inducing animmune response against the tumor antigen BFA5 (SEQ ID NO.: 6) in a hostcomprising administering to the host the isolated peptide of claim 79.82. A method for inducing an immune response against the tumor antigenBFA5 (SEQ ID NO.: 6) in a host comprising administering to the host thecomposition of claim 79.